Fitoterapia 102 (2015) 115–119
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Anti-cancer agents derived from solid-state fermented Antrodia camphorata mycelium I-Chuan Yen a,e, Chen-Wen Yao b, Mao-Tien Kuo c, Chen-Liang Chao d, Chien-Yi Pai b, Wen-Liang Chang e,* a
Graduate Institute of Medical Science, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu District, Taipei City 114, Taiwan, ROC Department of Pathology, Tri-Service General Hospital, No. 325, Sec. 2, Chenggong Road, Neihu District, Taipei City 114, Taiwan, ROC c LanTyng Biotech, Co., Ltd. Taipei, Taiwan, ROC d Graduate Institute of Life Science, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu District, Taipei City 114, Taiwan, ROC e School of Pharmacy, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu District, Taipei City 114, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 17 December 2014 Accepted in revised form 14 February 2015 Accepted 16 February 2015 Available online 23 February 2015 Keywords: Antrodia camphorata Ubiquinone derivative Antrocamol LT1 Antrocamol LT2 Antrocamol LT3 Cytotoxicity
a b s t r a c t Three new ubiquinone derivatives, antrocamol LT1, antrocamol LT2, and antrocamol LT3, along with two known compounds, were isolated from Antrodia camphorata (Polyporaceae) mycelium. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analyses. These ubiquinones exhibited selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 1.79 μΜ. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The indigenous Taiwanese medicinal mushroom Antrodia camphorata Chang and Chou, sp. nov. (Syn, Antrodia cinnamomea; Taiwanofungus camphoratus, Polyporaceae), locally known as “Niu Chang Chih”, is a parasitic fungus that grows in the inner cavity of Cinnamomum kanehirai Hay (Lauraceae) [1]. A. camphorata was historically used in Taiwan by the aborigines as a traditional prescription for the discomforts caused by excessive alcohol consumption or exhaustion [2]. The fruiting bodies have been used for the treatment of excessive food and alcohol consumption, drug intoxication, diarrhea, abdominal pain, hypertension, itchy skin and liver cancer in Chinese folk medicine [3]. However, the fruiting bodies have both an extremely slow growth rate ⁎ Corresponding author. Tel.: +886 2 87923100x18879; fax: +886 2 87923169. E-mail address: [email protected] (W.-L. Chang).
http://dx.doi.org/10.1016/j.fitote.2015.02.010 0367-326X/© 2015 Elsevier B.V. All rights reserved.
and a high growth specificity for the endangered C. kanehirai Hay tree. Thus, the mycelium of this fungus has become a substitute as a health food in Taiwan for the past few years [4]. Several triterpenoids, flavonoids, polysaccharides, organic acid, benzenoids, and ubiquinone derivatives have been identified from this fungus [4]. Both the fruiting bodies and mycelia of A. camphorata have been shown to exhibit a wide range of health-promoting benefits for hepatic, neurological, and cardiovascular systems. The extract of this fungus has been shown to inhibit inflammation, viral infection, oxidative stress, atherosclerosis, and the growth of several types of cancer cells [4–6]. Previous studies have shown that antroquinonol, a ubiquinone derivative found in the mycelium of A. camphorata, inhibits the growth of hepatocellular carcinoma (HCC) cells through the activation of AMPK and the inhibition of mTOR signaling [7], and the proliferation of NSCLC by altering PI3K/ mTOR proteins and miRNA expression profiles [8], and induces apoptosis and senescence in human pancreatic carcinoma cells
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[9]. The fungus has also been shown to inhibit iNOS activity and reduce oxidative stress through Nrf-2 activation [10–12]. In a search for anti-cancer agents from Chinese herbs, the ethanolic extract from A. camphorata mycelium was found to exhibit significant cytotoxic effects on lung, colon, hepatic, and prostate cancer cell lines. Hence this extract was reinvestigated, leading to the isolation and characterization of three new ubiquinone derivatives, antrocamol LT1 (1), antrocamol LT2 (2), and antrocamol LT3 (3), along with two known compounds. The cytotoxic effect of these compounds on five human cancer cells (CT26, A549, HepG2, PC3 and DU145) was also reported here. 2. Materials and methods 2.1. General experimental procedures Optical rotations were measured on a JASCO DIP 370 digital polarimeter. IR spectra were recorded in KBr disks on a PerkinElmer 983G spectrophotometer. UV spectra were obtained on a Shimadzu UV-160 spectrometer. 1H and 13C NMR spectra were determined with a Bruker AM-500 spectrometer using CDCl3, DMSO-d6 and MeOH-d4, with TMS as an internal standard. 2D NMR spectra were recorded using Bruker's standard pulse programs. HRESIMS and ESIMS were measured on a JEOL-JMS700 mass spectrometer and a Biosystems-Sciex API 3000 series triple-quadrupole mass spectrometer. 2.2. Materials Commercially available (S)-(+)-MTPACl and (R)-(−)MTPACl (Aldrish, no. 20445-33-4, [α]D + 137 ± 2; no. 39637-99-5, [α]D − 137 ± 2°) were used according to literature [13]. The dried mycelia of the A. camphorata were supplied by Lan-Tyng Co., Taipei, in December 2012. The fungus was authenticated by Prof. H.C. Lin, National Defense Medical Center, Taiwan, where a voucher specimen (NDMCP no. 1011201) has been deposited. 2.3. Preparation of the crude fungus extract The dried mycelia of the A. camphorata fungus (4.0 kg) were extracted with 95% ethanol (40 L × 2) at room temperature to yield a brown syrupy mass (LT-E; 909.0 g) after condensation under reduced pressure. This extract was suspended in H2O and then partitioned (1:1, v/v) with CH2Cl2 to obtain a CH2Cl2soluble fraction (LT-E-D; 405.2 g) and a water-soluble fraction (LT-E-W; 503.8 g). 2.4. Isolation of compounds The CH2Cl2-soluble fraction (20 g) was subjected to chromatography over silica gel and successively eluted with n-C6H14– CH2Cl2 (1:4), CH2Cl2 and CH2Cl2–MeOH (95:5) to generate five fractions. The second fraction (LT-E-D-2; 6.1 g) was applied to silica gel column chromatography, eluted with CH2Cl2–acetone (9:1) to yield 4,7-dimethoxy-5-methyl-1,3-benzodioxole (5), antrocamol LT2 (2; 7.0 mg), and antroquinonol (4; 59.0 mg). The third fraction (LT-E-D-3; 3.17 g) was applied to silica gel column chromatography, eluted with CH2Cl2–acetone (95:5) to yield
antrocamol LT1 (1; 268.8 mg) and antrocamol LT3 (3; 324.0 mg) (Fig. 1). 2.5. Compound 1 (antrocamol LT1) 2.5.1. Antrocamol LT1 (1) Colorless oil; [α]28 D +36.7 (c 0.3, MeOH); UV (MeOH)λmax (logε): 266 (4.0) nm; ESI-MS (positive) m/z (rel. int.%) 407 ([M+H]+, 12), 389 (66), 375 (31), 332 (42), 317 (50), 304 (80), 288 (100); IR (KBr) ν max: 3419, 2929, 1728, 1660, 1622, 1456, 1375, 1274, 1240, 1139, 1016, 970 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 407.2803 (calcd. for C24H39O5 [M+H]+ 407.2797). 2.6. Compound 2 (antrocamol LT2) 2.6.1. Antrocamol LT2 (2) Colorless oil; [α]28 D +98.7° (c 1.35, MeOH); UV (MeOH)λmax (logε): 262 (4.0) nm; ESI-MS (positive) m/z (rel. int.%) 449 ([M+H]+, 1), 431 (13), 403 (30), 389 (10), 371 (100); IR (KBr) ν max: 3507, 2937, 1749, 1671, 1630, 1455, 1366, 1236, 1141, 1012, 942 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 449.2910 (calcd. for C26H40O6 [M+H]+ 449.2903). 2.6.2. Compound 2a (S)-MTPACl (35.0 mg, 139 μmol) was added to a solution of 2 (4.5 mg, 10 μmol) in pyridine (346 μL). After stirring for 1 h at rt, the reaction mixture was diluted with hexane and quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with CH2Cl2 three times, and the combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by preparative TLC (hexane/EtOAc = 7:3) afforded (S)-MTPA ester 2a (3.1 mg, 4.67 μmol) in 46.6% yield as a yellow syrup: 1H NMR (400 MHz, CDCl3) δ 7.50–7.40 (m, 2H, phenyl group), 7.40–7.30 (m, 3H, phenyl group), 5.81 (dq, J = 6.8, 6.0 Hz, 1H, H-15), 5.71 (d, J = 3.2 Hz, 1H, H-4), 5.17 (m, 1H, H-12), 5.08 (m, 1H, H-8), 5.00 (d, J = 9.2 Hz, 1H, H-15) 3.97 (s, 3H, H-24), 3.65 (s, 3H, H-23), 3.50 (s, 3H, OCH3), 2.50 (dq, J = 6.8, 6.0 Hz, 1H, H-6), 2.41 (dd, J = 13.2, 8.0 Hz, 1H, H-14a), 2.18 (m, 1H, H-7a), 2.16 (dd, J = 13.2, 4.0 Hz, 1H, H-14b), 2.07 (s, 3H, acetate), 2.05 (m, 2H, H-11),1.96 (m, 2H, H-10), 1.94 (m, 1H, H-7b), 1.74 (s, 3H, H-19), 1.67 (s, 3H, H-18), 1.52 (s, 3H, H-21), 1.63 (s, 3H, H-20), 1.18 (d, J = 6.8 Hz, 3H, H-22). HRESIMS m/z 664.3233 [M+H]+ (calculated for C36H47F3O8 664.3223) [13]. 2.6.3. Compound 2b (R)-MTPACl (35.0 mg, 139 μmol) was added to a solution of 2 (4.5 mg, 10 μmol) in pyridine (346 μL). After stirring for 1 h at rt, the reaction mixture was diluted with hexane and quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with CH2Cl2 three times, and the combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by preparative TLC (hexane/EtOAc = 7:3) afforded (R)-MTPA ester 2b (3.0 mg, 4.53 μmol) in 45.2% yield as a yellow syrup: 1H NMR (400 MHz, CDCl3) δ 7.50–7.40 (m, 2H, phenyl group), 7.40– 7.30 (m, 3H, phenyl group), 5.81 (dq, J = 6.8, 6.0 Hz, 1H, H-15), 5.71 (d, J = 3.2 Hz, 1H, H-4), 5.18 (d, J = 9.2 Hz, 1H, H-15), 5.07 (m, 2H, H-8, 12), 3.97 (s, 3H, H-24), 3.65 (s, 3H, H-23), 3.50 (s, 3H, OCH3), 2.50 (dq, J = 6.8, 3.6 Hz, 1H, H-6), 2.32 (dd, J = 13.6,
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Fig. 1. Chemical structures of compounds 1–5.
8.0 Hz, 1H, H-14a), 2.13 (dd, J = 13.6, 6.4 Hz, 1H, H-14b), 2.18 (m, 1H, H-7a), 2.11 (m, 2H, H-11), 2.07 (s, 3H, acetate), 1.96 (m, 2H, H-10), 1.92 (m, 1H, H-7b), 1.74 (s, 3H, H-19), 1.72 (s, 3H, H-18), 1.54 (s, 3H, H-21), 1.51 (s, 3H, H-20), 1.18 (d, J = 6.8 Hz, 3H, H-22). HRESIMS m/z 664.3245 [M+H]+ (calculated for C36H47F3O8 664.3223) [13] (Table 1) (Fig. 2). 2.7. Compound 3 (antrocamol LT3) 2.7.1. Antrocamol LT3 (3) Colorless oil; [α]28 D + 27.7° (c 0.65, MeOH); UV (MeOH)λmax (logε): 263 (4.0) nm; ESI-MS m/z (rel. int.%) 407 ([M+H]+, 15), 389 (100), 375 (30); IR (KBr) ν max: 3419, 2972, 1732, 1668, 1622, 1456, 1377, 1238, 1141, 1074, 1018, 972 cm−1; 1H and 13C
Table 1 Comparison of MTPA ester data for 2(δ in ppm).
NMR, see Tables 2 and 3; HRESIMS m/z 407.2801 (calcd. for C24H39O5 [M+H]+ 407.2797). 2.8. Cell lines and culture The five human cancer cell lines (CT26, a colon carcinoma line; A549, a human lung carcinoma line; HepG2, a human liver carcinoma line; PC3 and DU-145, human prostate carcinoma lines) and MDCK (Madin–Darby canine kidney normal cell line) were obtained from the American Type Culture Collection (ATCC, USA). The cancer cell lines were maintained with F12 medium containing 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cultured cells were tested for mycoplasma infection using PCR screening methods before each experiment [16]. 2.9. Cytotoxicity analysis using the MTT assay
Position
(S)-MTPA ester
(R)-MTPA ester
Δδ = (δS − δR)
12 14a 14b 15 16 18 19 20
5.18 m 2.41 m 2.16 m 5.81 m 5.00 d 1.67 s 1.74 s 1.63 s
5.08 m 2.32 m 2.13 m 5.81 m 5.18 d 1.72 s 1.74 s 1.51 s
+0.10 +0.09 +0.03 0 −0.18 −0.15 0 +0.12
The viability of five human cancer cells (CT26, A549, HepG2, PC3 and DU-145) and MDCK cells treated with different concentrations of A. camphorata extracts, subfractions and isolates was evaluated using the MTT assay. Briefly, the cells were plated in 96-well plates (2 × 104 cells/well) and incubated overnight. The cells were treated with various concentrations of the alcohol extract, subfractions (0.01–200 μg/mL) or isolated compounds (0.0006–10 μg/mL, double dilution
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Fig. 2. Partial structures of MTPA ester of 2.
method) for 72 h. The medium was removed and cells were then incubated in 10 μL MTT solution (5 mg/mL) for 3.5 h at 37 °C. Lastly, the MTT solution was removed and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. 3. Results and discussion The CH2Cl2-soluble parts of the 95% ethanol extract of the dried mycelium of the fungus A. camphorata were subjected to silica gel column chromatography. This separation yielded three new ubiquinones: antrocamol LT1 (1), antrocamol LT2 (2) and antrocamol LT3 (3). Additionally, two known compounds, antroquinonol (4) and 4,7-dimethoxy-5-methyl-1,3benzodioxole (5) [14], were identified by comparison of their spectral data with literature values and were directly compared with authentic samples. The structures of the new compounds were elucidated as follows. Antrocamol LT1 (1) was obtained as a colorless oil and has a molecular formula of C24H38O5 (HRESIMS); it was 16 amu more than that of antroquinonol (4), which indicated the presence of an additional hydroxyl group. The UV and IR spectra were similar to those of antroquinonol (4), which indicated the presence of a ubiquinone moiety. The 1H NMR spectrum of 1 (Table 2) showed
Table 2 1 H spectroscopic data (CDCl3) for compounds 1–4 (δ in ppm and J in Hz). Position 1
2a
3
4 5 6
5.70 d (3.2) 1.84 m 2.48 dq (6.0, 6.8) 2.20 m, 1.92 m
4.31 d (2.8) 1.70 m 2.51 dq (9.6, 6.8) 2.21 m
7 8 10 11 12 14 15 16 18 19 20 21 22 23 24 a
4.33 d (2.8) 1.72 m 2.51 dq (11.6, 7.2) 2.33 dd (14.0, 7.2) 5.14 t (7.2) 2.06 m 2.17 m 5.15 t (7.2) 2.03 m, 2.14 m 4.35 dt (9.2, 4.0) 5.09 d (9.2) 1.68 s 1.66 s 1.64 s 1.61 s 1.12 d (7.2) 3.63 s 4.02 s
5.08 t (6.8) 1.96 m 2.11 m 5.18 t (6.4) 2.10 m 4.37 dt (8.0, 4.0) 5.10 d (8.0) 1.69 s 1.67 s 1.64 s 1.54 s 1.16 d (7.2) 3.64 s 3.97 s
Acetate group: δ 2.08 (q, OC_OCH3).
4
4.31 d (2.0) 1.74 m 2.51 dq (11.2, 6.9) 2.22 dd (7.4, 7.4) 5.14 t (6.8) 5.14 t (7.2) 2.02 m 2.03 m, 2.07 m 2.08 t 2.03 m, 2.07 m 5.08 t (6.4) 5.05 t (6.8) 2.64 d (4.8) 1.95 m, 2.03 m 5.56 m 1.95 m, 2.03 m 5.56 m 1.29 s 1.29 s 1.55 s 1.63 s 1.14 d (7.2) 3.63 s 4.04 s
5.05 t (6.8) 1.65 s 1.56 s 1.56 s 1.64 s 1.13 d (7.2) 3.60 s 4.02 s
proton signals for four primary methyl, four methylene, two methine, two methoxy, and three olefinic protons, similar to those of 4 except for the presence of two oxygenated methines instead of one in 4, with the additional one at (δ 4.35, dt, 1H) signal further downfield in 1. These results indicated the additional hydroxyl group link to C-15 of antroquinonol. The COSY45 spectrum showed correlations of the oxygenated methine proton (δ 4.35, dt, J = 4.0, 9.2 Hz, H-15) and an olefinic proton at δ 5.09 (1H, d, J = 9.2 Hz, H-16), as well as methylene protons at δ 2.03 and 2.14 (each 1H, m, H-14). An HMBC spectrum revealed a key coupling of H-15 to C-17, establishing also hydroxyl group at the C-15 position. The above data suggested 1 to be 4-hydroxy-2,3-dimethoxy-6-methyl-5(9-hydroxy-3,7,11trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone. The configuration of both C-8 and C-12 were the E form, as suggested by the δC of C-20 and C-21 at δC 15.94 and 16.18, respectively, in contrast to those of the Z configuration at approximately δC = 25.0 [15]. Antrocamol LT2 (2) was obtained as a colorless oil and has a molecular formula of C26H40O6 (HRESIMS). It was 42 amu more than that of 1, indicating the presence of an additional acetate Table 3 13 C NMR spectroscopic data (CDCl3) for compounds 1–4 (δ in ppm).a,b Position
1
2c
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
197.22 s 135.93 s 160.99 s 67.09 d 43.34 d 39.96 d 26.76 t 121.63 d 137.37 s 39.40 t 25.94 t 128.40 d 131.98 s 47.92 t 65.36 d 127.06 d 134.95 s 25.72 q 18.13 q 15.94 q 16.18 q 12.18 q 60.46 q 58.71 q
196.88 s 137.35 s 158.25 s 69.03 d 42.94 d 41.26 d 26.76 t 120.75 d 137.61 s 39.52 t 26.19 t 128.31 d 131.78 s 48.16 t 65.57 d 127.45 d 134.77 s 25.76 q 18.18 q 16.13 q 16.00 q 12.85 q 60.72 q 59.69 q
197.20 s 135.84 s 160.58 s 67.88 d 43.40 d 40.24 d 26.94 t 121.19 d 137.76 s 39.62 t 26.35 t 124.83 d 134.04 s 42.20 t 125.22 d 139.16 d 70.76 s 29.85 q 29.80 q 16.14 q 16.14 q 12.32 q 60.59 q 59.20 q
197.21 s 135.84 s 160.83 s 67.56 d 43.52 d 40.17 d 26.93 t 121.00 d 137.89 s 39.75 t 26.40 t 123.82 d 135.22 s 39.75 t 26.67 d 124.25 d 131.22 s 25.62 q 17.60 q 15.94 q 16.04 q 12.28 q 60.47 q 59.92 q
a
Multiplicities were obtained from DEPT experiments. Signals without Multiplicities were overlapped; chemical shifts with poorly resolved signals were assigned from COSY-45 or HMQC spectra. c Acetate group: δ 169.72 (s, C_O), 20.94 (q, CH3). b
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Table 4 Cytotoxic activity of crude extracts of Antrodia camphorata-derived, antrocamol LT1, antrocamol LT2 and antrocamol LT3 (μM)a. Samples
LT-E LT-E-D LT-E-D-3 1 2 3 4 5 Colchicineb Paclitaxelb a b
Cell lines MDCK
CT26
A549
HepG2
PC3
DU-145
N200 100 25 N10 N10 N10 N10 N10
10 1 0.1 0.17 1.79 0.03 0.21 N10
10 1 0.1 0.23 2.37 0.03 0.23 N10 0.27 0.00015
10 1 0.1 0.17 1.32 0.01 0.18 N10
1 0.1 0.1 0.14 1.54 0.02 0.03 N10 0.02 0.000094
1 0.1 0.1 0.14 2.03 0.17 0.03 N10 0.27 0.01394
The results shown here represent the mean (n = 3). Colchicine and paclitaxel were used as a positive control.
group. The NMR data of 2 closely resembled 1 except for an additional methyl of the acetate group at δ 2.08 (3H, s) and the oxygen-bearing methane proton that shifted downfield from δ 4.33 in 1 to δ 5.72 (1H, d, J = 3.2 Hz, H-4) in 2 indicated an acetate group at C-4 in 2. The additional acetate group at C-4 was based on the HMBC correlations observed between H-4 (δ 5.72) to the carbonyl of acetate (δ 169.72) and C-2 (δ 137.35). The above data indicated that 2 was 4-acetylantrocamol LT1, which we termed antrocamol LT2 (2). The absolute configuration at C-15 of this compound was determined to be R by the modified Mosher's method using MTPA ester [13]. Antrocamol LT3 (3) was obtained as a colorless oil and its molecular formula of C24H38O5 (HRESIMS) was the same as that of 1, which indicated the presence of an additional hydroxyl group. The NMR data of 3 were a close resemblance to 1 except that of H-13 (δ 2.64, 2H, br. d, J = 4.8 Hz). The signals from H-15 and 16 (overlapped at δ 5.56, 2H) were further downfield in 3, which indicated the additional hydroxyl group link to C-17 of antroquinonol. Therefore, compound 3 was elucidated as 4-hydroxy-2,3-dimethoxy-6-methyl-5(11hydroxy-3,7,11-trimethyl-dodeca-2,6,9-trienyl)-cyclohex2-enone and is termed antrocamol LT3 (3). These antrocamols were evaluated for their cytotoxicity against five human cancer cell lines (CT26, a colon carcinoma line, HepG2, a human liver carcinoma line, PC3 and DU-145, human prostate carcinoma lines) and MDCK (Madin-Darby canine kidney normal cell line) with an MTT assay. The antrocamols exhibited robust and selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 2.37 μM (Table 4). Notably, antrocamol LT3 (3) exhibited the most potent activity against these five cell lines with IC50 values of 0.03, 0.03, 0.01, 0.02 and 0.17 μM for the CT26, A549, HepG2, PC3 and DU-145 cell lines, respectively. The antrocamols, which are ubiquinone derivatives possessing a 4-hydroxy-2,3dimethoxy-6-methyl-cyclohex-2-enone ring, seemed to exhibit robust and selective cytotoxicity against the cancer cell lines, but the acetylation at C-4, e.g., antrocamol LT2 (2), reduced the activity of the antrocamols. The ethanol extract and antrocamols LT1–3 showed relatively low toxicity on a non-malignant cell
line (MDCK) with an IC50 of N 200 μg/mL and N10 μg/mL, respectively. Conflict of interest The authors declare no conflict of interest. Acknowledgment We are grateful to LanTyng Biotech, Co., Ltd. Taipei, Taiwan, R.O.C., for the support of these research materials. Appendix A. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.02.010. References [1] Chang TT, Chou WN. Mycol Res 1995;99(6):756. [2] Wu SH, Ryvarden L, Chang TT. Bot Bull Acad Sin 1997;38(4):273. [3] Tsai ZT, Liaw SL. The Use and the Effect of Ganoderma. Taichung, Taiwan: San Yun Press; 1985. [4] Geethangili M, Tzeng YM. Evid Based Complement Alternat Med 2011; 2011:1. [5] Ao ZH, Xu ZH, Lu ZM, Xu HY, Zhang XM, Dou WF. J Ethnopharmacol 2009; 121(2):194. [6] Geethangili M, Fang SH, Lai CH, Rao YK, Lien HM, Tzeng YM. Food Chem 2010;119(1):149. [7] Chiang PC, Lin SC, Pan SL, Kuo CH, Tsai IL, Kuo MT, et al. Biochem Pharmacol 2010;79:162. [8] Kumar VB, Yuan TC, Liou JW, Yang CJ, Sung PJ, Weng CF. Mutat Res 2011; 707:42. [9] Yu CC, Chiang PC, Lu PH, Kuo MT, Wen WC, Chen P, et al. J Nutr Biochem 2012;23(8):900. [10] Yang SS, Wang GJ, Wang SY, Lin YY, Kuo YH, Lee TH. Planta Med 2009; 75(5):512. [11] Tsai PY, Ka SM, Chao TK, Chang JM, Lin SH, Li CY, et al. Free Radic Biol Med 2011;50(11):1503. [12] Yang SM, Ka SM, Hua KF, Wu TH, Chuang YP, Lin YW, et al. Free Radic Biol Med 2013;61:285. [13] Ohtani I, Kusuni T, Kakisawa H. J Am Chem Soc 1991;113:4092–6. [14] Lien HM, Lin HW, Wang YJ, Chen LC, Yang DY, Lai YY, et al. Evid Based Complement Alternat Med 2009:1. [15] Pihlaja K, Kleinpeter E. Carbon-13 NMR chemical shifts in structural and stereochemical analysis. New York: VCH Publishers Inc.; 1994 231. [16] Uphoff CC, Drexler HG. Hum Cell 1999;12(4):229.
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Anti-cancer agents derived from solid-state fermented Antrodia camphorata mycelium I-Chuan Yen a,e, Chen-Wen Yao b, Mao-Tien Kuo c, Chen-Liang Chao d, Chien-Yi Pai b, Wen-Liang Chang e,* a
Graduate Institute of Medical Science, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu District, Taipei City 114, Taiwan, ROC Department of Pathology, Tri-Service General Hospital, No. 325, Sec. 2, Chenggong Road, Neihu District, Taipei City 114, Taiwan, ROC c LanTyng Biotech, Co., Ltd. Taipei, Taiwan, ROC d Graduate Institute of Life Science, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu District, Taipei City 114, Taiwan, ROC e School of Pharmacy, National Defense Medical Center, No. 161, Sec. 6, Minchuan East Road, Neihu District, Taipei City 114, Taiwan, ROC b
a r t i c l e
i n f o
Article history: Received 17 December 2014 Accepted in revised form 14 February 2015 Accepted 16 February 2015 Available online 23 February 2015 Keywords: Antrodia camphorata Ubiquinone derivative Antrocamol LT1 Antrocamol LT2 Antrocamol LT3 Cytotoxicity
a b s t r a c t Three new ubiquinone derivatives, antrocamol LT1, antrocamol LT2, and antrocamol LT3, along with two known compounds, were isolated from Antrodia camphorata (Polyporaceae) mycelium. The structures of these compounds were established on the basis of extensive 1D and 2D NMR spectroscopic analyses. These ubiquinones exhibited selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 1.79 μΜ. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The indigenous Taiwanese medicinal mushroom Antrodia camphorata Chang and Chou, sp. nov. (Syn, Antrodia cinnamomea; Taiwanofungus camphoratus, Polyporaceae), locally known as “Niu Chang Chih”, is a parasitic fungus that grows in the inner cavity of Cinnamomum kanehirai Hay (Lauraceae) [1]. A. camphorata was historically used in Taiwan by the aborigines as a traditional prescription for the discomforts caused by excessive alcohol consumption or exhaustion [2]. The fruiting bodies have been used for the treatment of excessive food and alcohol consumption, drug intoxication, diarrhea, abdominal pain, hypertension, itchy skin and liver cancer in Chinese folk medicine [3]. However, the fruiting bodies have both an extremely slow growth rate ⁎ Corresponding author. Tel.: +886 2 87923100x18879; fax: +886 2 87923169. E-mail address: [email protected] (W.-L. Chang).
http://dx.doi.org/10.1016/j.fitote.2015.02.010 0367-326X/© 2015 Elsevier B.V. All rights reserved.
and a high growth specificity for the endangered C. kanehirai Hay tree. Thus, the mycelium of this fungus has become a substitute as a health food in Taiwan for the past few years [4]. Several triterpenoids, flavonoids, polysaccharides, organic acid, benzenoids, and ubiquinone derivatives have been identified from this fungus [4]. Both the fruiting bodies and mycelia of A. camphorata have been shown to exhibit a wide range of health-promoting benefits for hepatic, neurological, and cardiovascular systems. The extract of this fungus has been shown to inhibit inflammation, viral infection, oxidative stress, atherosclerosis, and the growth of several types of cancer cells [4–6]. Previous studies have shown that antroquinonol, a ubiquinone derivative found in the mycelium of A. camphorata, inhibits the growth of hepatocellular carcinoma (HCC) cells through the activation of AMPK and the inhibition of mTOR signaling [7], and the proliferation of NSCLC by altering PI3K/ mTOR proteins and miRNA expression profiles [8], and induces apoptosis and senescence in human pancreatic carcinoma cells
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[9]. The fungus has also been shown to inhibit iNOS activity and reduce oxidative stress through Nrf-2 activation [10–12]. In a search for anti-cancer agents from Chinese herbs, the ethanolic extract from A. camphorata mycelium was found to exhibit significant cytotoxic effects on lung, colon, hepatic, and prostate cancer cell lines. Hence this extract was reinvestigated, leading to the isolation and characterization of three new ubiquinone derivatives, antrocamol LT1 (1), antrocamol LT2 (2), and antrocamol LT3 (3), along with two known compounds. The cytotoxic effect of these compounds on five human cancer cells (CT26, A549, HepG2, PC3 and DU145) was also reported here. 2. Materials and methods 2.1. General experimental procedures Optical rotations were measured on a JASCO DIP 370 digital polarimeter. IR spectra were recorded in KBr disks on a PerkinElmer 983G spectrophotometer. UV spectra were obtained on a Shimadzu UV-160 spectrometer. 1H and 13C NMR spectra were determined with a Bruker AM-500 spectrometer using CDCl3, DMSO-d6 and MeOH-d4, with TMS as an internal standard. 2D NMR spectra were recorded using Bruker's standard pulse programs. HRESIMS and ESIMS were measured on a JEOL-JMS700 mass spectrometer and a Biosystems-Sciex API 3000 series triple-quadrupole mass spectrometer. 2.2. Materials Commercially available (S)-(+)-MTPACl and (R)-(−)MTPACl (Aldrish, no. 20445-33-4, [α]D + 137 ± 2; no. 39637-99-5, [α]D − 137 ± 2°) were used according to literature [13]. The dried mycelia of the A. camphorata were supplied by Lan-Tyng Co., Taipei, in December 2012. The fungus was authenticated by Prof. H.C. Lin, National Defense Medical Center, Taiwan, where a voucher specimen (NDMCP no. 1011201) has been deposited. 2.3. Preparation of the crude fungus extract The dried mycelia of the A. camphorata fungus (4.0 kg) were extracted with 95% ethanol (40 L × 2) at room temperature to yield a brown syrupy mass (LT-E; 909.0 g) after condensation under reduced pressure. This extract was suspended in H2O and then partitioned (1:1, v/v) with CH2Cl2 to obtain a CH2Cl2soluble fraction (LT-E-D; 405.2 g) and a water-soluble fraction (LT-E-W; 503.8 g). 2.4. Isolation of compounds The CH2Cl2-soluble fraction (20 g) was subjected to chromatography over silica gel and successively eluted with n-C6H14– CH2Cl2 (1:4), CH2Cl2 and CH2Cl2–MeOH (95:5) to generate five fractions. The second fraction (LT-E-D-2; 6.1 g) was applied to silica gel column chromatography, eluted with CH2Cl2–acetone (9:1) to yield 4,7-dimethoxy-5-methyl-1,3-benzodioxole (5), antrocamol LT2 (2; 7.0 mg), and antroquinonol (4; 59.0 mg). The third fraction (LT-E-D-3; 3.17 g) was applied to silica gel column chromatography, eluted with CH2Cl2–acetone (95:5) to yield
antrocamol LT1 (1; 268.8 mg) and antrocamol LT3 (3; 324.0 mg) (Fig. 1). 2.5. Compound 1 (antrocamol LT1) 2.5.1. Antrocamol LT1 (1) Colorless oil; [α]28 D +36.7 (c 0.3, MeOH); UV (MeOH)λmax (logε): 266 (4.0) nm; ESI-MS (positive) m/z (rel. int.%) 407 ([M+H]+, 12), 389 (66), 375 (31), 332 (42), 317 (50), 304 (80), 288 (100); IR (KBr) ν max: 3419, 2929, 1728, 1660, 1622, 1456, 1375, 1274, 1240, 1139, 1016, 970 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 407.2803 (calcd. for C24H39O5 [M+H]+ 407.2797). 2.6. Compound 2 (antrocamol LT2) 2.6.1. Antrocamol LT2 (2) Colorless oil; [α]28 D +98.7° (c 1.35, MeOH); UV (MeOH)λmax (logε): 262 (4.0) nm; ESI-MS (positive) m/z (rel. int.%) 449 ([M+H]+, 1), 431 (13), 403 (30), 389 (10), 371 (100); IR (KBr) ν max: 3507, 2937, 1749, 1671, 1630, 1455, 1366, 1236, 1141, 1012, 942 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 449.2910 (calcd. for C26H40O6 [M+H]+ 449.2903). 2.6.2. Compound 2a (S)-MTPACl (35.0 mg, 139 μmol) was added to a solution of 2 (4.5 mg, 10 μmol) in pyridine (346 μL). After stirring for 1 h at rt, the reaction mixture was diluted with hexane and quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with CH2Cl2 three times, and the combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by preparative TLC (hexane/EtOAc = 7:3) afforded (S)-MTPA ester 2a (3.1 mg, 4.67 μmol) in 46.6% yield as a yellow syrup: 1H NMR (400 MHz, CDCl3) δ 7.50–7.40 (m, 2H, phenyl group), 7.40–7.30 (m, 3H, phenyl group), 5.81 (dq, J = 6.8, 6.0 Hz, 1H, H-15), 5.71 (d, J = 3.2 Hz, 1H, H-4), 5.17 (m, 1H, H-12), 5.08 (m, 1H, H-8), 5.00 (d, J = 9.2 Hz, 1H, H-15) 3.97 (s, 3H, H-24), 3.65 (s, 3H, H-23), 3.50 (s, 3H, OCH3), 2.50 (dq, J = 6.8, 6.0 Hz, 1H, H-6), 2.41 (dd, J = 13.2, 8.0 Hz, 1H, H-14a), 2.18 (m, 1H, H-7a), 2.16 (dd, J = 13.2, 4.0 Hz, 1H, H-14b), 2.07 (s, 3H, acetate), 2.05 (m, 2H, H-11),1.96 (m, 2H, H-10), 1.94 (m, 1H, H-7b), 1.74 (s, 3H, H-19), 1.67 (s, 3H, H-18), 1.52 (s, 3H, H-21), 1.63 (s, 3H, H-20), 1.18 (d, J = 6.8 Hz, 3H, H-22). HRESIMS m/z 664.3233 [M+H]+ (calculated for C36H47F3O8 664.3223) [13]. 2.6.3. Compound 2b (R)-MTPACl (35.0 mg, 139 μmol) was added to a solution of 2 (4.5 mg, 10 μmol) in pyridine (346 μL). After stirring for 1 h at rt, the reaction mixture was diluted with hexane and quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with CH2Cl2 three times, and the combined organic extracts were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by preparative TLC (hexane/EtOAc = 7:3) afforded (R)-MTPA ester 2b (3.0 mg, 4.53 μmol) in 45.2% yield as a yellow syrup: 1H NMR (400 MHz, CDCl3) δ 7.50–7.40 (m, 2H, phenyl group), 7.40– 7.30 (m, 3H, phenyl group), 5.81 (dq, J = 6.8, 6.0 Hz, 1H, H-15), 5.71 (d, J = 3.2 Hz, 1H, H-4), 5.18 (d, J = 9.2 Hz, 1H, H-15), 5.07 (m, 2H, H-8, 12), 3.97 (s, 3H, H-24), 3.65 (s, 3H, H-23), 3.50 (s, 3H, OCH3), 2.50 (dq, J = 6.8, 3.6 Hz, 1H, H-6), 2.32 (dd, J = 13.6,
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Fig. 1. Chemical structures of compounds 1–5.
8.0 Hz, 1H, H-14a), 2.13 (dd, J = 13.6, 6.4 Hz, 1H, H-14b), 2.18 (m, 1H, H-7a), 2.11 (m, 2H, H-11), 2.07 (s, 3H, acetate), 1.96 (m, 2H, H-10), 1.92 (m, 1H, H-7b), 1.74 (s, 3H, H-19), 1.72 (s, 3H, H-18), 1.54 (s, 3H, H-21), 1.51 (s, 3H, H-20), 1.18 (d, J = 6.8 Hz, 3H, H-22). HRESIMS m/z 664.3245 [M+H]+ (calculated for C36H47F3O8 664.3223) [13] (Table 1) (Fig. 2). 2.7. Compound 3 (antrocamol LT3) 2.7.1. Antrocamol LT3 (3) Colorless oil; [α]28 D + 27.7° (c 0.65, MeOH); UV (MeOH)λmax (logε): 263 (4.0) nm; ESI-MS m/z (rel. int.%) 407 ([M+H]+, 15), 389 (100), 375 (30); IR (KBr) ν max: 3419, 2972, 1732, 1668, 1622, 1456, 1377, 1238, 1141, 1074, 1018, 972 cm−1; 1H and 13C
Table 1 Comparison of MTPA ester data for 2(δ in ppm).
NMR, see Tables 2 and 3; HRESIMS m/z 407.2801 (calcd. for C24H39O5 [M+H]+ 407.2797). 2.8. Cell lines and culture The five human cancer cell lines (CT26, a colon carcinoma line; A549, a human lung carcinoma line; HepG2, a human liver carcinoma line; PC3 and DU-145, human prostate carcinoma lines) and MDCK (Madin–Darby canine kidney normal cell line) were obtained from the American Type Culture Collection (ATCC, USA). The cancer cell lines were maintained with F12 medium containing 10% FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cultured cells were tested for mycoplasma infection using PCR screening methods before each experiment [16]. 2.9. Cytotoxicity analysis using the MTT assay
Position
(S)-MTPA ester
(R)-MTPA ester
Δδ = (δS − δR)
12 14a 14b 15 16 18 19 20
5.18 m 2.41 m 2.16 m 5.81 m 5.00 d 1.67 s 1.74 s 1.63 s
5.08 m 2.32 m 2.13 m 5.81 m 5.18 d 1.72 s 1.74 s 1.51 s
+0.10 +0.09 +0.03 0 −0.18 −0.15 0 +0.12
The viability of five human cancer cells (CT26, A549, HepG2, PC3 and DU-145) and MDCK cells treated with different concentrations of A. camphorata extracts, subfractions and isolates was evaluated using the MTT assay. Briefly, the cells were plated in 96-well plates (2 × 104 cells/well) and incubated overnight. The cells were treated with various concentrations of the alcohol extract, subfractions (0.01–200 μg/mL) or isolated compounds (0.0006–10 μg/mL, double dilution
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Fig. 2. Partial structures of MTPA ester of 2.
method) for 72 h. The medium was removed and cells were then incubated in 10 μL MTT solution (5 mg/mL) for 3.5 h at 37 °C. Lastly, the MTT solution was removed and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader. 3. Results and discussion The CH2Cl2-soluble parts of the 95% ethanol extract of the dried mycelium of the fungus A. camphorata were subjected to silica gel column chromatography. This separation yielded three new ubiquinones: antrocamol LT1 (1), antrocamol LT2 (2) and antrocamol LT3 (3). Additionally, two known compounds, antroquinonol (4) and 4,7-dimethoxy-5-methyl-1,3benzodioxole (5) [14], were identified by comparison of their spectral data with literature values and were directly compared with authentic samples. The structures of the new compounds were elucidated as follows. Antrocamol LT1 (1) was obtained as a colorless oil and has a molecular formula of C24H38O5 (HRESIMS); it was 16 amu more than that of antroquinonol (4), which indicated the presence of an additional hydroxyl group. The UV and IR spectra were similar to those of antroquinonol (4), which indicated the presence of a ubiquinone moiety. The 1H NMR spectrum of 1 (Table 2) showed
Table 2 1 H spectroscopic data (CDCl3) for compounds 1–4 (δ in ppm and J in Hz). Position 1
2a
3
4 5 6
5.70 d (3.2) 1.84 m 2.48 dq (6.0, 6.8) 2.20 m, 1.92 m
4.31 d (2.8) 1.70 m 2.51 dq (9.6, 6.8) 2.21 m
7 8 10 11 12 14 15 16 18 19 20 21 22 23 24 a
4.33 d (2.8) 1.72 m 2.51 dq (11.6, 7.2) 2.33 dd (14.0, 7.2) 5.14 t (7.2) 2.06 m 2.17 m 5.15 t (7.2) 2.03 m, 2.14 m 4.35 dt (9.2, 4.0) 5.09 d (9.2) 1.68 s 1.66 s 1.64 s 1.61 s 1.12 d (7.2) 3.63 s 4.02 s
5.08 t (6.8) 1.96 m 2.11 m 5.18 t (6.4) 2.10 m 4.37 dt (8.0, 4.0) 5.10 d (8.0) 1.69 s 1.67 s 1.64 s 1.54 s 1.16 d (7.2) 3.64 s 3.97 s
Acetate group: δ 2.08 (q, OC_OCH3).
4
4.31 d (2.0) 1.74 m 2.51 dq (11.2, 6.9) 2.22 dd (7.4, 7.4) 5.14 t (6.8) 5.14 t (7.2) 2.02 m 2.03 m, 2.07 m 2.08 t 2.03 m, 2.07 m 5.08 t (6.4) 5.05 t (6.8) 2.64 d (4.8) 1.95 m, 2.03 m 5.56 m 1.95 m, 2.03 m 5.56 m 1.29 s 1.29 s 1.55 s 1.63 s 1.14 d (7.2) 3.63 s 4.04 s
5.05 t (6.8) 1.65 s 1.56 s 1.56 s 1.64 s 1.13 d (7.2) 3.60 s 4.02 s
proton signals for four primary methyl, four methylene, two methine, two methoxy, and three olefinic protons, similar to those of 4 except for the presence of two oxygenated methines instead of one in 4, with the additional one at (δ 4.35, dt, 1H) signal further downfield in 1. These results indicated the additional hydroxyl group link to C-15 of antroquinonol. The COSY45 spectrum showed correlations of the oxygenated methine proton (δ 4.35, dt, J = 4.0, 9.2 Hz, H-15) and an olefinic proton at δ 5.09 (1H, d, J = 9.2 Hz, H-16), as well as methylene protons at δ 2.03 and 2.14 (each 1H, m, H-14). An HMBC spectrum revealed a key coupling of H-15 to C-17, establishing also hydroxyl group at the C-15 position. The above data suggested 1 to be 4-hydroxy-2,3-dimethoxy-6-methyl-5(9-hydroxy-3,7,11trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone. The configuration of both C-8 and C-12 were the E form, as suggested by the δC of C-20 and C-21 at δC 15.94 and 16.18, respectively, in contrast to those of the Z configuration at approximately δC = 25.0 [15]. Antrocamol LT2 (2) was obtained as a colorless oil and has a molecular formula of C26H40O6 (HRESIMS). It was 42 amu more than that of 1, indicating the presence of an additional acetate Table 3 13 C NMR spectroscopic data (CDCl3) for compounds 1–4 (δ in ppm).a,b Position
1
2c
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
197.22 s 135.93 s 160.99 s 67.09 d 43.34 d 39.96 d 26.76 t 121.63 d 137.37 s 39.40 t 25.94 t 128.40 d 131.98 s 47.92 t 65.36 d 127.06 d 134.95 s 25.72 q 18.13 q 15.94 q 16.18 q 12.18 q 60.46 q 58.71 q
196.88 s 137.35 s 158.25 s 69.03 d 42.94 d 41.26 d 26.76 t 120.75 d 137.61 s 39.52 t 26.19 t 128.31 d 131.78 s 48.16 t 65.57 d 127.45 d 134.77 s 25.76 q 18.18 q 16.13 q 16.00 q 12.85 q 60.72 q 59.69 q
197.20 s 135.84 s 160.58 s 67.88 d 43.40 d 40.24 d 26.94 t 121.19 d 137.76 s 39.62 t 26.35 t 124.83 d 134.04 s 42.20 t 125.22 d 139.16 d 70.76 s 29.85 q 29.80 q 16.14 q 16.14 q 12.32 q 60.59 q 59.20 q
197.21 s 135.84 s 160.83 s 67.56 d 43.52 d 40.17 d 26.93 t 121.00 d 137.89 s 39.75 t 26.40 t 123.82 d 135.22 s 39.75 t 26.67 d 124.25 d 131.22 s 25.62 q 17.60 q 15.94 q 16.04 q 12.28 q 60.47 q 59.92 q
a
Multiplicities were obtained from DEPT experiments. Signals without Multiplicities were overlapped; chemical shifts with poorly resolved signals were assigned from COSY-45 or HMQC spectra. c Acetate group: δ 169.72 (s, C_O), 20.94 (q, CH3). b
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Table 4 Cytotoxic activity of crude extracts of Antrodia camphorata-derived, antrocamol LT1, antrocamol LT2 and antrocamol LT3 (μM)a. Samples
LT-E LT-E-D LT-E-D-3 1 2 3 4 5 Colchicineb Paclitaxelb a b
Cell lines MDCK
CT26
A549
HepG2
PC3
DU-145
N200 100 25 N10 N10 N10 N10 N10
10 1 0.1 0.17 1.79 0.03 0.21 N10
10 1 0.1 0.23 2.37 0.03 0.23 N10 0.27 0.00015
10 1 0.1 0.17 1.32 0.01 0.18 N10
1 0.1 0.1 0.14 1.54 0.02 0.03 N10 0.02 0.000094
1 0.1 0.1 0.14 2.03 0.17 0.03 N10 0.27 0.01394
The results shown here represent the mean (n = 3). Colchicine and paclitaxel were used as a positive control.
group. The NMR data of 2 closely resembled 1 except for an additional methyl of the acetate group at δ 2.08 (3H, s) and the oxygen-bearing methane proton that shifted downfield from δ 4.33 in 1 to δ 5.72 (1H, d, J = 3.2 Hz, H-4) in 2 indicated an acetate group at C-4 in 2. The additional acetate group at C-4 was based on the HMBC correlations observed between H-4 (δ 5.72) to the carbonyl of acetate (δ 169.72) and C-2 (δ 137.35). The above data indicated that 2 was 4-acetylantrocamol LT1, which we termed antrocamol LT2 (2). The absolute configuration at C-15 of this compound was determined to be R by the modified Mosher's method using MTPA ester [13]. Antrocamol LT3 (3) was obtained as a colorless oil and its molecular formula of C24H38O5 (HRESIMS) was the same as that of 1, which indicated the presence of an additional hydroxyl group. The NMR data of 3 were a close resemblance to 1 except that of H-13 (δ 2.64, 2H, br. d, J = 4.8 Hz). The signals from H-15 and 16 (overlapped at δ 5.56, 2H) were further downfield in 3, which indicated the additional hydroxyl group link to C-17 of antroquinonol. Therefore, compound 3 was elucidated as 4-hydroxy-2,3-dimethoxy-6-methyl-5(11hydroxy-3,7,11-trimethyl-dodeca-2,6,9-trienyl)-cyclohex2-enone and is termed antrocamol LT3 (3). These antrocamols were evaluated for their cytotoxicity against five human cancer cell lines (CT26, a colon carcinoma line, HepG2, a human liver carcinoma line, PC3 and DU-145, human prostate carcinoma lines) and MDCK (Madin-Darby canine kidney normal cell line) with an MTT assay. The antrocamols exhibited robust and selective cytotoxicities against five human cancer cell lines (CT26, A549, HepG2, PC3 and DU-145) with IC50 values ranging from 0.01 to 2.37 μM (Table 4). Notably, antrocamol LT3 (3) exhibited the most potent activity against these five cell lines with IC50 values of 0.03, 0.03, 0.01, 0.02 and 0.17 μM for the CT26, A549, HepG2, PC3 and DU-145 cell lines, respectively. The antrocamols, which are ubiquinone derivatives possessing a 4-hydroxy-2,3dimethoxy-6-methyl-cyclohex-2-enone ring, seemed to exhibit robust and selective cytotoxicity against the cancer cell lines, but the acetylation at C-4, e.g., antrocamol LT2 (2), reduced the activity of the antrocamols. The ethanol extract and antrocamols LT1–3 showed relatively low toxicity on a non-malignant cell
line (MDCK) with an IC50 of N 200 μg/mL and N10 μg/mL, respectively. Conflict of interest The authors declare no conflict of interest. Acknowledgment We are grateful to LanTyng Biotech, Co., Ltd. Taipei, Taiwan, R.O.C., for the support of these research materials. Appendix A. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.02.010. References [1] Chang TT, Chou WN. Mycol Res 1995;99(6):756. [2] Wu SH, Ryvarden L, Chang TT. Bot Bull Acad Sin 1997;38(4):273. [3] Tsai ZT, Liaw SL. The Use and the Effect of Ganoderma. Taichung, Taiwan: San Yun Press; 1985. [4] Geethangili M, Tzeng YM. Evid Based Complement Alternat Med 2011; 2011:1. [5] Ao ZH, Xu ZH, Lu ZM, Xu HY, Zhang XM, Dou WF. J Ethnopharmacol 2009; 121(2):194. [6] Geethangili M, Fang SH, Lai CH, Rao YK, Lien HM, Tzeng YM. Food Chem 2010;119(1):149. [7] Chiang PC, Lin SC, Pan SL, Kuo CH, Tsai IL, Kuo MT, et al. Biochem Pharmacol 2010;79:162. [8] Kumar VB, Yuan TC, Liou JW, Yang CJ, Sung PJ, Weng CF. Mutat Res 2011; 707:42. [9] Yu CC, Chiang PC, Lu PH, Kuo MT, Wen WC, Chen P, et al. J Nutr Biochem 2012;23(8):900. [10] Yang SS, Wang GJ, Wang SY, Lin YY, Kuo YH, Lee TH. Planta Med 2009; 75(5):512. [11] Tsai PY, Ka SM, Chao TK, Chang JM, Lin SH, Li CY, et al. Free Radic Biol Med 2011;50(11):1503. [12] Yang SM, Ka SM, Hua KF, Wu TH, Chuang YP, Lin YW, et al. Free Radic Biol Med 2013;61:285. [13] Ohtani I, Kusuni T, Kakisawa H. J Am Chem Soc 1991;113:4092–6. [14] Lien HM, Lin HW, Wang YJ, Chen LC, Yang DY, Lai YY, et al. Evid Based Complement Alternat Med 2009:1. [15] Pihlaja K, Kleinpeter E. Carbon-13 NMR chemical shifts in structural and stereochemical analysis. New York: VCH Publishers Inc.; 1994 231. [16] Uphoff CC, Drexler HG. Hum Cell 1999;12(4):229.
